Two Forms of Astrocyte Calcium Excitability Have Distinct Effects on NMDA Receptor-Mediated Slow Inward Currents in Pyramidal Neurons

The Journal of Neuroscience, June 25, 2008 • 28(26):6659–6663 • 6659

Brief Communications Two Forms of Astrocyte Calcium Excitability Have Distinct Effects on NMDA Receptor-Mediated Slow Inward Currents in Pyramidal Neurons

Eiji Shigetomi,1,2 David N. Bowser,3 Michael V. Sofroniew,2 and Baljit S. Khakh1,2,3 Departments of 1Physiology and 2Neurobiology, David Geffen School of Medicine, University of California, Los Angeles, California 90095-1751, and 3Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom

2ϩ Astrocytes display excitability in the form of intracellular calcium concentration ([Ca ]i ) increases, but the signaling impact of these for 2ϩ neurons remains debated and controversial. A key unresolved issue is whether astrocyte [Ca ]i elevations impact neurons or not. Here we report that in the CA1 region of the hippocampus, agonists of native P2Y1 and PAR-1 receptors, which are preferentially expressed in 2ϩ astrocytes, equally elevated [Ca ]i levels without affecting the passive membrane properties of pyramidal neurons. However, under conditions chosen to isolate NMDA receptor responses, we found that activation of PAR-1 receptors led to the appearance of NMDA receptor-mediated slow inward currents (SICs) in pyramidal neurons. In stark contrast, activation of P2Y1 receptors was ineffective in 2ϩ this regard. The PAR-1 receptor-mediated increased SICs were abolished by several strategies that selectively impaired astrocyte [Ca ]i 2ϩ excitability and function. Our studies therefore indicate that evoked astrocyte [Ca ]i transients are not a binary signal for interactions with neurons, and that astrocytes result in neuronal NMDA receptor-mediated SICs only when appropriately excited. The data thus provide a basis to rationalize recent contradictory data on astrocyte–neuron interactions. Key words: astrocyte; calcium; SIC; gliotransmitter; astrocytic glutamate release; glia

Introduction cellular Ca 2ϩ release (Newman, 2003a; Parri and Crunelli, 2003). 2ϩ Astrocytes, a type of glial cell, are found in the brain, and their Moreover, it is now well established that astrocyte [Ca ]i in- close spatial relationship to neurons is well documented (Araque creases trigger release of transmitters into the extracellular space. et al., 2001; Haydon, 2001). In addition to their important house- The evidence in favor of this is derived from a variety of ap- keeping roles (Kofuji and Newman, 2004), increasing evidence proaches, including biochemical, amperometric, and capacitance now suggests that astrocytes also actively participate in neuronal methods, as well as single-vesicle imaging (Araque et al., 2000; function (Haydon, 2001; Newman, 2003b; Fields, 2004; Fields Krzan et al., 2003; Bezzi et al., 2004; Evanko et al., 2004; Kreft et and Burnstock, 2006). al., 2004; Zhang et al., 2004a,b; Bowser and Khakh, 2007a; Jaiswal Astrocytes do not fire action potentials. However, they display et al., 2007). One of the transmitters known to be released from ϩ spontaneous and pharmacologically evoked intracellular Ca 2 astrocytes is glutamate. 2ϩ 2ϩ concentration ([Ca ]i) increases, which represent a form of as- The realization that astrocytes display [Ca ]i elevations and trocyte excitability. Since their initial discovery (Cornell-Bell et that they display calcium-triggered glutamate release raises the 2ϩ al., 1990) in astrocyte cultures, [Ca ]i increases have been ex- possibility that astrocytic glutamate could affect neurons. This tensively studied for in vitro preparations of brain slices (Haydon, issue has been addressed in the past, and we restrict ourselves here 2001; Parri et al., 2001; Newman, 2003b; Fields, 2004), and have to the most pertinent experiments for the CA1 region of the also been recorded from astrocytes in vivo with two-photon cal- hippocampus. In one study, NMDA receptor-mediated slow in- 2ϩ cium imaging from the cortex (Hirase et al., 2004). Thus [Ca ]i ward currents (SICs) were recorded from CA1 pyramidal neu- elevations appear to be a widespread and prevalent feature of rons when (S)-3,5-dihydroxyphenylglycine (DHPG) was used to astrocytes, can be triggered by neuronal activity, and are known activate metabotropic glutamate receptors on astrocytes and thus to occur independently of neuronal activity resulting from intra- 2ϩ trigger [Ca ]i elevations (Fellin et al., 2004). However, a recent study has questioned the use of DHPG because of its attendant Received April 20, 2008; revised May 15, 2008; accepted May 16, 2008. actions on neurons (Fiacco et al., 2007). Instead these authors This work was supported partly by the Medical Research Council, the Whitehall Foundation, and the Stein/ Oppenheimer Endowment Award (B.S.K.) and an Uehara Memorial Foundation Fellowship (Japan) (E.S.). used the creative approach of using an exogenous MrgA1 recep- 2ϩ Correspondence should be addressed to Baljit S. Khakh, Department of Physiology, University of California, Los tor selectively expressed in astrocytes to elevate [Ca ]i within Angeles, 10833 LeConte Avenue, 53-263 CHS, Los Angeles, CA 90095-1751. E-mail: [email protected]. 2ϩ them. They found that when astrocyte[Ca ]i levels were ele- D. N. Bowser’s present address: Howard Florey Institute, University of Melbourne, Parkville, Victoria 3010, vated by activating MrgA1 receptors, this did not result in the Australia. DOI:10.1523/JNEUROSCI.1717-08.2008 appearance of SICs in pyramidal neurons (Fiacco et al., 2007). Copyright © 2008 Society for Neuroscience 0270-6474/08/286659-05$15.00/0 Thus, the starkly contrasting conclusions between groups (Fellin 6660 • J. Neurosci., June 25, 2008 • 28(26):6659–6663 Shigetomi et al. • Astrocyte–Neuron Interactions et al., 2004; Fiacco et al., 2007) leaves open the question of 2ϩ whether astrocyte [Ca ]i elevations impact neurons or not (Tritsch and Bergles, 2007). In the present study, we returned to 2ϩ the issue of whether astrocyte [Ca ]i elevations can cause the appearance of SICs on pyramidal neurons. Materials and Methods Preparation of brain slices and electrophysiological recording. The methods used have been described previously (Khakh et al., 2003; Bowser and Khakh, 2004). Briefly, young [postnatal day 11 (P11)–P19] C57 mice were killed in accordance with institutional procedures. In some cases, GFAP-green fluorescent protein (GFP) mice were used, and these were available from previous work (Garcia et al., 2004). Coronal slices of hippocampus (200 or 300 ␮m) were cut (model 1000 or 3000 Plus Vi- bratome) and submerged at room temperature in artificial CSF (aCSF) comprising the following (in mM): 126 NaCl, 2.5 KCl, 1.3 MgCl2,10 D-glucose, 2.4 CaCl2, 1.24 NaH2PO4, and 26 NaHCO3 saturated with 95% O2 and 5% CO2. Experiments were performed with potassium gluconate or chloride-based internal solution, comprising the following (in Ϫ mM): 120 K gluconate (or Cl ), 10 KCl, 1 MgCl2, 0.03 CaCl2, 0.1 EGTA, 1 ATP, 0.2 GTP, 10 HEPES, and 4 glucose, pH 7.25. The resistance of the pipettes was ϳ4–9M⍀. Cells were visualized with infrared optics (Luigs and Neumann) on an upright microscope (Olympus BX50). We used bath application of agonists. Inward currents were recorded at Ϫ60 mV using pCLAMP 9 software, Multiclamp 900A amplifier, and Digidata 1322A (Molecular Devices). Spontaneous EPSCs were recorded in the presence of bicuculline (10 ␮M) and D-(Ϫ)-2-amino-5-phosphono- pentanoic acid (D-AP5; 10 ␮M). Slow inward currents (Fellin et al., 2004) mediated by extrasynaptic NMDA receptors were recorded in low-Mg 2ϩ buffers (0.13 mM) in the presence of bicuculline (10 ␮M), TTX (1 ␮M), and 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (CNQX; 10 ␮M). Astro- cytes were patched with 4–9 M⍀ electrodes filled with an intracellular solution comprising 123 CsCl, 1 MgSO , 10 HEPES, 1 ATP, 0.2 GTP, pH 4 2ϩ 7.35, and 100 ␮M Alexa Fluor 488 (referred to as Alexa 488 in the text), Figure1. Astrocyte[Ca ]i transientsevokedbyactivationofP2Y1 andPAR-1receptors.A, M Representative images of the stratum radiatum region of the hippocampus with astrocytes and BAPTA (10 m ) as indicated in the text (Jourdain et al., 2007). ϩ 2ϩ 2 ␮ Confocal imaging of astrocyte Ca activity and dialysis. Brain slices showingincreasesin[Ca ]i duringactivationofPAR-1receptorswithTFLLRpeptide(30 M). were loaded at room temperature in the dark with 5 ␮M Fluo-4 AM Arrowheads point to astrocytes showing intracellular calcium transients. B, Exemplar traces 2ϩ (Invitrogen) in aCSF for 60 min, then transferred to dye-free aCSF for at PAR-1- and P2Y1-evoked [Ca ]i transients. C, D, Pooled data for experiments such as those in least 30 min before experimentation to allow for cleavage of the AM ester A.Thegraylinesareindividualcells,andthesolidlinesaretheaveragesfromallcells.Activation 2ϩ group (Porter and McCarthy, 1996). Live astrocytes were predominantly of both P2Y1 and PAR-1 receptors evoked increases in astrocyte [Ca ]i levels. E, The average 2ϩ loaded with the fluorescent dye with these conditions. This was con- normalized traces for [Ca ]i transients evoked by P2Y1 and PAR-1 receptor activation; note firmed by simultaneous acquisition of infrared-differential interference that there was a latency for the PAR-1-mediated responses in relation to those mediated by contrast (IR-DIC) images of astrocytes in the same area. Cells and slices P2Y1 receptors, and their duration was longer. were imaged using an Olympus Fluoview 300 laser-scanning confocal microscope with a 300 mW argon laser, at Ͻ10% power. Emitted green fluorescence was collected through a 515 long-pass filter. Fluoview soft- vesicle exocytosis in culture (Bowser and Khakh, 2007a). PAR-1 ware was used for image acquisition. For BAPTA and Alexa 488 dialysis, receptors are known to be preferentially expressed within astro- the astrocytes were held at Ϫ80 mV, and depolarized to 0 mV for 100 ms cytes of the hippocampus (Weinstein et al., 1995; Junge et al., every 30 s to monitor access resistance. The optimal dialysis time was 2004; Lee et al., 2007). Thus, P2Y1 and PAR-1 receptors are useful empirically determined to be 15 min after patching. Astrocyte dialysis targets to increase astrocyte [Ca 2ϩ] levels, and determine ␮ i with Alexa 488 was measured from confocal stacks for 90 m depth from whether this affects neurons in brain slices. To this end, we the surface of the brain slices for the stratum radiatum region within started by loading astrocytes in hippocampal slices with Fluo-4 150–200 ␮m of the CA1 pyramidal cell layer. We counted all astrocytes in AM and focused on the stratum radiatum region. We found that this layer that were visibly fluorescent (see Results). ␤ ␮ the P2Y1 receptor agonist ADP S (30 or 100 M), and PAR-1 Chemicals. All chemicals used were from Tocris Bioscience, Sigma, or ␮ Invitrogen. The chemicals used were CNQX, D-AP5, and adenosine 5Ј- receptor agonists thrombin (10 U/ml) and TFLLR-NH2 (30 M) 2ϩ ␤-thio diphosphate (ADP␤S). elevated [Ca ]i in astrocytes within the stratum radiatum re- Analysis. Synaptic currents were analyzed using MiniAnalysis program gion. This is readily seen in the representative images and traces 5.6.22 (Synaptosoft), Strathclyde Electrophysiology Software (John in Figure 1, A and B, and Figure 1, C and D, shows 23 and 14 Dempster, University of Strathclyde, Glasgow, UK), pCLAMP9 (Molec- superimposed sweeps with the average data emphasized in thick ular Devices), Origin 6.1 (OriginLab), and GraphPad Instat 3.06 for 2ϩ lines. Interestingly, the PAR-1 agonist-evoked [Ca ]i transients Windows. Confocal and epifluorescence imaging data were analyzed us- were slower to develop compared with the P2Y1 receptor agonist ing ImageJ and CorelDraw 12. Data in the text and graphs are shown as ones (Fig. 1E), and although the evoked transients displayed mean Ϯ SEM from n determinations as indicated. ⌬ Ϯ Ϯ equal peaks ( F/F of 0.8 0.1 and 0.6 0.1 for P2Y1 and PAR-1 Results receptors; n ϭ 23 and 14), the PAR-1-mediated events lasted P2Y1 and PAR-1 receptor agonists excite astrocytes significantly longer (the widths of the events at 25% of the peak Ϯ Ϯ We recently demonstrated that agonists of P2Y1 and PAR-1 re- height were 36 4 and 77 9 s, respectively, for P2Y1 and PAR-1 2ϩ ϭ ceptors both elevate astrocyte [Ca ]i levels and triggered single- transients; n 23 and 14). We next used fura-2 AM-based imag- Shigetomi et al. • Astrocyte–Neuron Interactions J. Neurosci., June 25, 2008 • 28(26):6659–6663 • 6661

Ϫ43 Ϯ 1 pA; n ϭ 316). Both the SICs and EP- SCs reported here for 200- to 300-␮m-thick mouse coronal hippocampal slices were ap- proximately twice as fast (rise and decay) as those reported previously from 300- to 400- ␮m-thick rat transverse hippocampal slices (Fellin et al., 2004), but nonetheless readily dis- tinguishable from each other (Fig. 2A). To test whether SICs were indeed caused by astrocyte release of glutamate acting on extrasynaptic NMDA receptors, we tested the actions of the NR2B antagonist ifenprodil (10 ␮M; n ϭ 8), which selectively blocks SICs mediated by astrocytes (Fellin et al., 2004). The presence of ifenprodil, or APV, reduced or abolished spontaneous SICs (n ϭ 8) (Fig. 2C), suggesting that SICs were mediated by extrasynaptic NMDA receptors (Fellin et al., 2004). ␤ The P2Y1 agonist ADP S produced no effect on the holding current of pyramidal neurons (ϳ1 pA change; n ϭ 19) and no effect on SICs (1.8 Ϯ 0.3 SICs/min before ADP␤S and 1.5 Ϯ 0.3 SICs/min during ADP␤S; n ϭ 8). 2ϩ Figure 2. Effect of astrocyte [Ca ]i excitation on glutamatergic SICs onto pyramidal neurons. A, Representative (top) Thus, the effect of P2Y1 receptor activation was andaverage(bottom)tracesforSICsandEPSCsrecordedfromapyramidalneuron(averagedataarepresentedinthetext). minimal, suggesting that elevation of astrocyte B,ActivationofPAR-1receptorsledtotheappearanceofSICs.C,ThespontaneousSICswerereducedorabolishedwithAPV ϩ [Ca 2 ] levels has little postsynaptic conse- and ifenprodil, and in slices pretreated with FA and BAPTA-AM. D, PAR-1 agonist-evoked SICs were abolished in the i presence of ifenprodil or in slices pretreated with FA or BAPTA-AM. quences for CA1 pyramidal neurons. Throm- ␮ bin and TFLLR-NH2 (10 U/ml and 30 M), which activate PAR-1 receptors and elevate as- 2ϩ 2ϩ ing to estimate the change in astrocyte [Ca ]i. We estimate that trocyte [Ca ]i levels, produced no effect on the holding current ϳ Ͼ ϭ activation of P2Y1 and PAR-1 receptors elevated astrocyte of pyramidal neurons ( 1 pA change; p 0.05; n 25). How- 2ϩ ϳ ␮ ϳ [Ca ]i levels to 0.5 M from a resting level of 50 nM (for a ever, PAR-1 receptor activation increased the frequency of SICs Ϯ specific set of experiments, the peaks were 407 69 nM for P2Y1 mediated by extrasynaptic NMDA receptors (Fig. 2) (12/12 cells). activation, 305 Ϯ 56 nM for PAR-1 activation). Thus, within the To further explore the hypothesis that the PAR-1 receptor-

stratum radiatum region of the hippocampus, agonists of P2Y1 evoked SICs are of astrocyte origin, we used fluoroacetate (FA; 2ϩ ␮ and PAR-1 receptors increase astrocyte [Ca ]i levels to the slices were pretreated with 10 M FA for 2 h) to selectively impair range expected to trigger glutamate release (Parpura and Hay- astrocyte function in brain slices (Fonnum et al., 1997; Gordon et don, 2000; Kreft et al., 2004). In contrast to their robust effect on al., 2005) and BAPTA-AM (Liu et al., 2004) to chelate astrocyte 2ϩ ␮ astrocyte [Ca ]i levels (Fig. 1), in supplemental Figure 1 (avail- intracellular calcium (200 M BAPTA-AM for 1.5 h followed by able at www.jneurosci.org as supplemental material) we present 30 min at room temperature). We loaded Fluo-4 AM together

several controls indicating that P2Y1 and PAR-1 receptor agonists with BAPTA-AM, as well as into slices treated with FA, to verify 2ϩ do not alter pyramidal neuron excitability. that the [Ca ]i transients were abolished in astrocytes. They were: we did not observe any spontaneous P2Y1- or PAR-1- 2ϩ Effects of astrocyte excitation on astrocyte–pyramidal evoked [Ca ]i transients in slices treated with FA or BAPTA- neuron interactions AM. Additionally, in slices treated with FA or BAPTA-AM, we ϭ We exploited the finding that P2Y1 and PAR-1 receptor activa- observed no SICs (Fig. 2C)(n 5 and 5) and no PAR-1 receptor- 2ϩ ϭ tion evoked significant astrocyte [Ca ]i transients in slices (Fig. evoked SICs (Fig. 2D)(n 5 and 5) in pyramidal neurons. 2ϩ 1), as well as exocytosis (Bowser and Khakh, 2007a) to determine Overall, these findings suggest that [Ca ]i transients in astro- whether these affect CA1 pyramidal neurons. Electron micros- cytes evoked with P2Y1 agonist (Fig. 1) result in minor postsyn- 2ϩ copy has shown that astrocytes are intimately associated with aptic effects on pyramidal neurons. In contrast, [Ca ]i tran- neurons in the CA1 region (Bushong et al., 2002). We monitored sients evoked with PAR-1 receptor agonists (Fig. 1) result in slow inward currents (Fellin et al., 2004; Perea and Araque, 2005) postsynaptic extrasynaptic NMDA responses (Fellin et al., 2004; mediated by extrasynaptic NMDA receptors (Fig. 2), which are Perea and Araque, 2005) in pyramidal neurons (Fig. 2). We per- distinct from EPSCs. The SICs were recorded at a holding poten- formed controls, which together show that BAPTA-AM and FA tial of Ϫ60 mV in the presence of TTX, bicuculline, and CNQX did not adversely affect synaptic currents or the passive and active (1, 10, and 10 ␮M, respectively) in extracellular buffers with 10- membrane properties of pyramidal neurons (supplemental Fig. 2ϩ fold-reduced Mg (0.13 mM). Based on previous work (Fellin et 1, available at www.jneurosci.org as supplemental material). al., 2004), we identified the SICs because of their slow rise times (10–90% rise time ϭ 46 Ϯ 18 ms), slow decay times (␶ ϭ 196 Ϯ Astrocyte dialysis with BAPTA impairs PAR-1 92 ms), and low frequency (0.03 Ϯ 0.005 Hz; the amplitude was receptor-evoked SICs Ϫ29 Ϯ 1 pA; n ϭ 132). In contrast to SICs, synaptic EPSCs were We next sought to dialyze astrocytes in brain slices with BAPTA faster (10–90% rise time ϭ 1.2 Ϯ 0.06 ms; decay ␶ ϭ 6.6 Ϯ 1.4 through the patch pipette to further determine whether the spon- ms) and more frequent (0.66 Ϯ 0.09 Hz; the amplitude was taneous and PAR-1 receptor-evoked SICs have an astrocytic ori- 6662 • J. Neurosci., June 25, 2008 • 28(26):6659–6663 Shigetomi et al. • Astrocyte–Neuron Interactions gin. We started by studying the spread of Al- exa 488 dye to establish conditions needed for complete dialysis. By patching single stratum radiatum astrocytes within ϳ150–200 ␮m of the CA1 pyramidal cell layer, we determined that 15 min of dialysis resulted in dialysis of ϳ46 astrocytes with Alexa 488 (100 ␮M). We next repeated these experiments with 10 mM BAPTA as well as Alexa 488 in the pipette solution, and found that a similar number of astrocytes were coupled (ϳ70) (Fig. 3B,E). We also counted the number of GFP-positive astrocytes from confocal stacks from brain slices from trans- genic mice expressing GFP in astrocytes. We found that these areas contained ϳ73 GFP- positive astrocytes. Because we could dialyze ϳ70 astrocytes by patching one single astro- Figure 3. Dialysis of astrocytes with BAPTA via the patch pipette abolishes the PAR-1 agonist-evoked SICs in pyramidal cyte with BAPTA and Alexa 488, and because neurons. A, Diagram of the hippocampus; the green box shows the stratum radiatum area that we focused on for astrocyte ϳ73 GFP-positive astrocytes were found in dialysis and recording. B, A flattened confocal stack shows numerous astrocytes that were dialyzed with Alexa Fluor 488 after the same area, we conclude that our dialysis patchingasingleastrocyte(thepatchpipetteisseenenteringfromtheright).C,AsinBbutfordialysiswithAlexaFluor488and protocol results in BAPTA loading into most 10 mM BAPTA; note that many astrocytes were dialyzed. D, GFP-positive astrocytes from the stratum radiatum region of brain of the available astrocytes. slices harvested from GFAP-GFP mice. Note that many astrocytes expressed GFP. E, A summary bar graph of the numbers of Using the aforementioned protocol for astrocytes that were dialyzed with Alexa Fluor 488 and BAPTA, and the number of astrocytes counted from GFAP-GFP mice. F, Top,AnIR-DICimageofthesurfaceofahippocampalslicefortheregionshowninA.Astrocyteswithinϳ150–200␮mofthe astrocyte dialysis, we then recorded from py- ␮ pyramidal layer were patched and dialyzed with Alexa Fluor 488 and BAPTA. Bottom, A fluorescence image of the same slice. ramidal neurons within 150–200 m of the Notethatastrocytesweredialyzedevenonthesurfaceoftheslice.Thetotalnumberofastrocytesdialyzedwithin90␮mdepth patched astrocyte to determine whether ispresentedinE.G,Top,ArepresentativetraceofPAR-1agonist-evokedSICsarrivingontopyramidalneuronsinaslicewithout PAR-1 receptor-evoked SICs occurred. In BAPTAdialysisintoastrocytes.Bottom,Thesameexperiment,butinthiscaseforslicesinwhichastrocytesnearthepyramidal control slices, in which the astrocytes had not neuron had been dialyzed with BAPTA. Note that in this case, there were no SICs. The asterisks indicate SICs. Average data are been dialyzed with BAPTA, we recorded ro- presented in the text. DG, Dentate gyrus; SP, stratum pyramidale; wt, wild type. bust PAR-1 receptor-evoked SICs (7.3 Ϯ 1.5 per neuron) (Fig. 3G)(n ϭ 9/15 cells; 6 cells showed no re- PAR-1 receptors triggers glutamate release (Bowser and Khakh, sponse). However, in slices in which the astrocytes had been di- 2007a) and activation of extrasynaptic NMDA receptors on py- alyzed with BAPTA, we recorded no spontaneous or PAR-1 ramidal neurons. Ϯ ϭ receptor-evoked SICs (0.1 0.1 per neuron) (Fig. 3D,G)(n Interestingly, our data show that agonists of P2Y1 and PAR-1 2ϩ 8/8 cells). Thus, we conclude that dialysis of the calcium chelator receptors equally increased astrocyte [Ca ]i levels, but that the 2ϩ BAPTA selectively into astrocytes impairs PAR-1 receptor- PAR-1-mediated [Ca ]i transients were of longer duration. evoked SICs arriving onto pyramidal neurons, providing strong Might the differences in neuronal SIC generation resulting from support for our hypothesis that elevation of astrocyte intracellu- P2Y1 and PAR-1 receptor activation be attributable to the fact 2ϩ 2ϩ lar [Ca ]i by PAR-1 receptor agonists causes release of gluta- that [Ca ]i remains elevated for a longer time after PAR-1 acti- mate that activates NMDA receptors on pyramidal neurons. The vation? We cannot rule this possibility out. However, new meth- data presented in supplemental Figure 2 (available at www. ods will be needed to probe this systematically and reliably. This is jneurosci.org as supplemental material) strongly suggest that because rapidly applying and removing P2Y1 and PAR-1 agonists P2Y1 and PAR-1 receptor agonists do not release astrocyte trans- in multicellular brain slice preparations is not possible, and so a mitter via volume-sensitive channels, simply because no such pharmacological approach to control the duration of agonist- 2ϩ channels were detected. Our data favor the view that PAR-1 re- evoked [Ca ]i transient is problematic. These problems are ceptor activation leads to exocytosis of neurotransmitter- compounded by the fact that P2Y1 receptor agonists are ATP containing vesicles (Bowser and Khakh, 2007a). analogues and are thus labile. Moreover, agonists of PAR-1 receptors are peptides with slow penetration into slices. In the fu- 2ϩ Discussion ture, it may be useful to precisely control astrocyte [Ca ]i levels Using agonists of two natively expressed receptors (P2Y1 and by light-evoked activation of channelrhodopsin 2, which allows PAR-1) within astrocytes, we show that (1) both agonists elevate significant calcium flux into cells (Nagel et al., 2003). This chan- 2ϩ [Ca ]i within astrocytes to an equal level, and (2) only activa- nel has been used to excite neurons (Boyden et al., 2005), but tion of the PAR-1 receptor leads to neuronal consequences in the because of its significant divalent cation permeability, it should form of NMDA receptor-mediated SICs. This difference in the prove useful for optical calcium excitation of astrocytes in trans- neuronal impact of activating P2Y1 and PAR-1 receptors may genic mice (Nagel et al., 2003). This possibility will be systemat- relate to the modes of exocytosis that these agonists trigger ically explored in future work. (Bowser and Khakh, 2007a), or the spatial location of the gluta- How do our studies relate to other recent work for the stratum mate release sites. For instance, we have shown that only PAR-1 radiatum of the hippocampus? The fact that we could measure receptor-mediated astrocyte excitation results in full fusion spontaneous SICs and increases in their frequency by exciting astro- forms of exocytosis (Bowser and Khakh, 2007a), and that ATP- cytes is consistent with previous work with DHPG, and flash photol- evoked exocytosis largely, if not solely, affects other astrocytes ysis of caged calcium (Fellin et al., 2004). We note that the osmolarity (Bowser and Khakh, 2007b). In contrast, activation of astrocyte of our solutions was constant, and so the recent proposition that Shigetomi et al. • Astrocyte–Neuron Interactions J. Neurosci., June 25, 2008 • 28(26):6659–6663 • 6663

SICs are attributable to hypo-osmotic stimuli (Fiacco et al., 2007) Gordon GR, Baimoukhametova DV, Hewitt SA, Rajapaksha WR, Fisher TE, does not apply. However, our findings do not detract from the ap- Bains JS (2005) Norepinephrine triggers release of glial ATP to increase proach of activating exogenously expressed MrgA1 receptors that postsynaptic efficacy. Nat Neurosci 8:1078–1086. Haydon PG (2001) GLIA: listening and talking to the synapse. Nat Rev Neu- failed to show SICs on pyramidal neurons (Fiacco et al., 2007). In- rosci 2:185–193. deed, Fiacco et al. (2007) argued that the method of elevating astro- Hirase H, Qian L, Bartho´ P, Buzsa´ki G (2004) Calcium dynamics of cortical 2ϩ cyte [Ca ]i levels is key to whether glutamate release can occur. astrocytic networks in vivo. PLoS Biol 2:E96; 494–499. However, their approach assumes that MrgA1 receptors produce Jaiswal JK, Fix M, Takano T, Nedergaard M, Simon SM (2007) Resolving 2ϩ [Ca ]i signals like those for native receptors. This was the case in vesicle fusion from lysis to monitor calcium-triggered lysosomal exocy- terms of the peak change in fluorescence for a calcium indicator dye, tosis in astrocytes. Proc Natl Acad Sci USA 104:14151–14156. but our experiments show that this linear extrapolation to a down- Jourdain P, Bergersen LH, Bhaukaurally K, Bezzi P, Santello M, Domercq M, stream effect (i.e., signaling to neurons) is not straightforward. Of Matute C, Tonello F, Gundersen V, Volterra A (2007) Glutamate exocytosis from astrocytes controls synaptic strength. Nat Neurosci relevance is the novel observation reported here that two natively 2ϩ 10:331–339. expressed receptors produced similar peak changes in [Ca ]i but Junge CE, Lee CJ, Hubbard KB, Zhang Z, Olson JJ, Hepler JR, Brat DJ, produced distinct effects on neurons. On the other hand, both re- Traynelis SF (2004) Protease-activated receptor-1 in human brain: lo- ceptors are known to trigger exocytosis (Bowser and Khakh, 2007a). calization and functional expression in astrocytes. Exp Neurol 2ϩ Our data suggest that when astrocyte [Ca ]i levels are appropriately 188:94–103. elevated, by activating native PAR-1 receptors, glutamate release Khakh BS, Gittermann D, Cockayne DA, Jones A (2003) ATP modulation of does occur with neuronal consequences (Lee and Haydon, 2007). excitatory synapses onto interneurons. J Neurosci 23:7426–7437. Overall, our data underscore the need for caution when interpreting Kofuji P, Newman EA (2004) Potassium buffering in the central nervous 2ϩ system. Neuroscience 129:1045–1056. astrocyte [Ca ]i imaging data, especially with exogenous receptors Kreft M, Stenovec M, Rupnik M, Grilc S, Krzan M, Potokar M, Pangrsic T, whose targeting in spatially distinct aspects of astrocytes is not doc- Haydon PG, Zorec R (2004) Properties of Ca(2ϩ)-dependent exocyto- umented. More generally, our data show that the relationship be- sis in cultured astrocytes. Glia 46:437–445. 2ϩ tween astrocyte [Ca ]i excitability and neuronal impact is not Krzan M, Stenovec M, Kreft M, Pangrsic T, Grilc S, Haydon PG, Zorec R binary. (2003) Calcium-dependent exocytosis of atrial natriuretic peptide from astrocytes. J Neurosci 23:1580–1583. References Lee CJ, Mannaioni G, Yuan H, Woo DH, Gingrich MB, Traynelis SF (2007) Araque A, Li N, Doyle RT, Haydon PG (2000) SNARE protein-dependent Astrocytic control of synaptic NMDA receptors. J Physiol (Lond) glutamate release from astrocytes. J Neurosci 20:666–673. 581:1057–1081. Araque A, Carmignoto G, Haydon PG (2001) Dynamic signalling between Lee SY, Haydon PG (2007) Astrocytic glutamate targets NMDA receptors. astrocytes and neurons. Annu Rev Physiol 63:795–813. J Physiol (Lond) 581:887–888. Bezzi P, Gundersen V, Galbete JL, Seifert G, Steinha¨user C, Pilati E, Volterra Liu QS, Xu Q, Arcuino G, Kang J, Nedergaard M (2004) Astrocyte-mediated A (2004) Astrocytes contain a vesicular compartment that is competent activation of neuronal kainate receptors. Proc Natl Acad Sci USA for regulated exocytosis of glutamate. Nat Neurosci 7:613–620. 101:3172–3177. Bowser DN, Khakh BS (2004) ATP excites interneurons and astrocytes to Nagel G, Szellas T, Huhn W, Kateriya S, Adeishvili N, Berthold P, Ollig D, increase synaptic inhibition in neuronal networks. J Neurosci Hegemann P, Bamberg E (2003) Channelrhodopsin-2, a directly light- 24:8606–8620. gated cation-selective membrane channel. Proc Natl Acad Sci USA Bowser DN, Khakh BS (2007a) Two forms of single vesicle astrocyte exocy- 100:13940–13945. tosis imaged with total internal reflection fluorescence microscopy. Proc Newman EA (2003a) Glial cell inhibition of neurons by release of ATP. Natl Acad Sci USA 104:4212–4217. J Neurosci 23:1659–1666. Bowser DN, Khakh BS (2007b) Vesicular ATP is the predominant cause of Newman EA (2003b) New roles for astrocytes: regulation of synaptic trans- intercellular calcium waves in astrocytes. J Gen Physiol 129:485–491. mission. Trends Neurosci 26:536–542. Boyden ES, Zhang F, Bamberg E, Nagel G, Deisseroth K (2005) Millisecond- Parpura V, Haydon PG (2000) Physiological astrocytic calcium levels stim- timescale, genetically targeted optical control of neural activity. Nat Neu- ulate glutamate release to modulate adjacent neurons. Proc Natl Acad Sci rosci 8:1263–1268. USA 97:8629–8634. Bushong EA, Martone ME, Jones YZ, Ellisman MH (2002) Protoplasmic Parri HR, Crunelli V (2003) The role of Ca2ϩ in the generation of sponta- astrocytes in CA1 stratum radiatum occupy separate anatomical domains. neous astrocytic Ca2ϩ oscillations. Neuroscience 120:979–992. J Neurosci 22:183–192. Parri HR, Gould TM, Crunelli V (2001) Spontaneous astrocytic Ca2ϩ os- Cornell-Bell AH, Finkbeiner SM, Cooper MS, Smith SJ (1990) Glutamate cillations in situ drive NMDAR-mediated neuronal excitation. Nat Neu- induces calcium waves in cultured astrocytes: long-range glial signaling. rosci 4:803–812. Science 247:470–473. Evanko DS, Zhang Q, Zorec R, Haydon PG (2004) Defining pathways of Perea G, Araque A (2005) Properties of synaptically evoked astrocyte cal- loss and secretion of chemical messengers from astrocytes. Glia cium signal reveal synaptic information processing by astrocytes. J Neu- 47:233–240. rosci 25:2192–2203. Fellin T, Pascual O, Gobbo S, Pozzan T, Haydon PG, Carmignoto G (2004) Porter JT, McCarthy KD (1996) Hippocampal astrocytes in situ respond to Neuronal synchrony mediated by astrocytic glutamate through activation glutamate released from synaptic terminals. J Neurosci 16:5073–5081. of extrasynaptic NMDA receptors. Neuron 43:729–743. Tritsch NX, Bergles DE (2007) Defining the role of astrocytes in neuro- Fiacco TA, Agulhon C, Taves SR, Petravicz J, Casper KB, Dong X, Chen J, modulation. Neuron 54:497–500. McCarthy KD (2007) Selective stimulation of astrocyte calcium in situ Weinstein JR, Gold SJ, Cunningham DD, Gall CM (1995) Cellular localiza- does not affect neuronal excitatory synaptic activity. Neuron 54:611–626. tion of thrombin receptor mRNA in rat brain: expression by mesence- Fields RD (2004) The other half of the brain. Sci Am 290:54–61. phalic dopaminergic neurons and codistribution with prothrombin Fields RD, Burnstock G (2006) Purinergic signalling in neuron-glia interac- mRNA. J Neurosci 15:2906–2919. tions. Nat Rev Neurosci 7:423–436. Zhang Q, Pangrsic T, Kreft M, Krzan M, Li N, Sul JY, Halassa M, Van Bock- Fonnum F, Johnsen A, Hassel B (1997) Use of fluorocitrate and fluoroac- staele E, Zorec R, Haydon PG (2004a) Fusion-related release of gluta- etate in the study of brain metabolism. Glia 21:106–113. mate from astrocytes. J Biol Chem 279:12724–12733. Garcia AD, Doan NB, Imura T, Bush TG, Sofroniew MV (2004) GFAP- Zhang Q, Fukuda M, Van Bockstaele E, Pascual O, Haydon PG (2004b) expressing progenitors are the principal source of constitutive neurogen- Synaptotagmin IV regulates glial glutamate release. Proc Natl Acad Sci esis in adult mouse forebrain. Nat Neurosci 7:1233–1241. USA 101:9441–9446.